1.1 Field of the Invention
The present invention provides amphiphilic prodrugs comprising a therapeutic compound conjugated to an PEG-oligomer/polymer and methods for using said prodrugs to enable oral drug delivery and/or delivery of drugs across the blood brain barrier (BBB).
1.2 Related Art
The following is a discussion of art related to the present invention.
The American Cancer Society estimates that 16,800 people will be diagnosed with primary tumors of the central nervous system (CNS) in the United States in 1999, and that 13,100 of these will eventually die of their disease (American Cancer Society, 1999). Primary CNS tumors are among the most intractable cancers to treat. While such tumors rarely metastasize, their anatomic location causes a high incidence of morbidity and mortality resulting from compression of surrounding tissue as well as destruction of tissue invaded by such tumors. Additionally, CNS tumor cells often migrate away from the tumor to other locations in the brain. These migratory cells eventually form recurrent tumors. Multiple CNS tumors can also result from metastases of non-CNS neoplasmas.
Standard treatment for CNS tumors includes surgery and radiation therapy. However, with regard to surgery, complete surgical resection is often not possible. Normal brain tissue adjacent to the tumor is often critical to the survival or the quality of life of the patient. Consequently, survival of patients treated by surgery is low. The problems associated with radiation therapy are widely known in the art.
Systemic chemotherapy, would be a valuable therapeutic option for CNS tumors due to its ability to target microscopic deposits of tumor cells, including cells missed by radiation and surgery. However, the inability to get such agents across the BBB has proven to be a significant limitation to the use of chemotherapy in the CNS. Many agents with activity against tumors do not cross the BBB to enter the brain parenchyma. Further, although many proven antineoplastic agents, including etoposide, will accumulate in brain tumors (Kiya, Uozumi et al. 1992), the concentration of these drugs decreases rapidly with distance from the tumor (Donelli, Zucchetti et al. 1992). In order to achieve effective treatment of the whole brain and expose microdeposits of tumor cells to drug, it would be desirable to have means for delivering therapeutic concentrations of anti-cancer drugs to the entire volume of the brain.
The brain is equipped with a barrier system which must be traversed to permit therapeutic drug delivery to the CNS. This brain barrier system has two major components: the choroid plexus and the blood-brain barrier (BBB). The choroid plexus separates cerebrospinal fluid (CSF) from the bloodstream, and the BBB separates brain interstitial fluid (ISF) from blood.
The BBB has about 1000 times more surface area than the choroid plexus and is the primary obstacle to delivery of therapeutic compounds to the CNS. The BBB acts as a selective partition, regulating the exchange of substances, between the CNS and the peripheral circulation. The primary structure of the BBB is the brain capillary endothelial wall. The tight junctions of brain capillary endothelial cells prevent circulating compounds from reaching the brain ISF by the paracellular route. Furthermore, recent work suggests the existence of a separate physiological barrier at the level of the basal lamina (Kroll et al. 1998). Other unique characteristics of the BBB include lack of intracellular fenestrations and pinocytic vesicles and a net negative charge on the luminal surface of the endothelium (Kroll et al. 1998).
The mechanisms by which substances traverse the BBB may generally be divided into active and passive transport mechanisms. Lipophilic molecules readily traverse the BBB by passive transport or diffusion through the endothelial plasma membranes, while hydrophilic molecules typically require an active transport system. Diffusion of many therapeutic compounds, across the BBB is also inhibited by size.
Many currently existing drug substances are unable to overcome these structural and metabolic barriers to enter the BBB in sufficient quantities to be efficacious. There is therefore a need for pharmaceutical compounds which can enable penetration of anti-cancer drugs through the BBB in sufficient amounts and at sufficient rates to be efficacious. Furthermore, many substances which, in theory, should be able to cross the BBB due to their lipophilicity, are not suitable for parenteral or oral delivery in the absence of potentially allergenic formulation ingredients. There is a need in the art for prodrugs which increase the solubility of drugs, preferably resulting in amphiphilic drugs which are orally available, soluble in the bloodstream, and which improve the ability of such drugs to enter the CNS. Conversely, there is a need in the art for prodrugs which increase the lipophilicity of hydrophilic drugs to produce amphiphilic prodrugs. Moreover, it is desirable that such amphiphilic prodrugs are hydrolyzed in vivo to release the active parent compound.
Many attempts have been made in the art to deliver therapeutic compounds to the CNS with varying levels of success. Such attempts can generally be grouped into two categories: invasive and pharmacological.
Invasive delivery strategies include, for example, mechanical procedures, such as implantation of an intraventricular catheter, followed by pharmaceutical infusion into the ventricular compartment. Aside from general considerations relating to the invasiveness of mechanical procedures, a major difficulty with mechanical approaches is the lack of drug distribution. For example, injection of drugs into the CSF compartment commonly results in very little distribution beyond the surface of the brain. This lack of distribution is due in part to rapid exportation of drugs to the peripheral circulation.
Another invasive strategy for delivering therapeutic compounds to the CNS is by intracartoid infusion of highly concentrated osmotically active substances, such as mannitol or arabinose. Their high local concentration causes shrinkages of the brain capillary endothelial cells, resulting in a transient opening of the tight junctions which enable molecules to traverse the BBB. Such procedures have considerable toxic effects, including inflammation, encephalitis, etc. Furthermore, such procedures are not selective: the opening of the tight junctions of the BBB permits many undesirable substances to cross the BBB along with the therapeutically beneficial molecule. For a recent review of osmotic opening and other invasive means for traversing the BBB, see Kroll, Neurosurgery, Vol. 42, No. 5, May 1998.
There is therefore a need in the art for means for selectively enabling therapeutic agents, such as peptides, to cross the BBB in a controlled manner which permits accumulation of sufficient quantities of the therapeutic in the brain to induce the desired therapeutic effect.
The present inventors have surprisingly discovered that conjugation of small amphiphilic polymers to drugs, such as etoposide, solves many of the aforementioned difficulties. This approach relies on rational oligomer design using a hydrophobic component plus a hydrophilic component to balance the physiochemical properties of the parent molecule. By varying the molecular weight of the hydrophobic and hydrophilic components of the oligomer and/or the molecular weight of the amphiphilic portion of the oligomer, the overall physiochemical profile of the conjugated molecule can be systematically adjusted to produce the desired degree of amphiphilicity with concomitant alterations in solubility and pharmacokinetics.
The etoposide prodrugs of the present invention can effectively cross the blood-brain barrier. Based on the empirical data presented herein and on the results of our prior research described in U.S. patent application Ser. No. 09/134,803, entitled xe2x80x9cBlood-Brain Barrier Therapeutics,xe2x80x9d the etoposide prodrugs are predicted to have improved oral bioavailability as well. These two elements, coupled with the ability to control the rate of hydrolysis of the prodrug from the drug compound, facilitate the use of chronic dosing regimens for the treatment of cancer of the CNS and other malignancies.
The present inventors have surprisingly discovered that covalent modification of a hydrophobic drug with the oligomers of the present invention counteracts the hydrophobic nature of the parent compound and vastly improves its ability to penetrate the blood-brain barrier. Furthermore, the inventors have discovered that conjugation of a lipophilic parent drug, e.g., etoposide, with the oligomers of the present invention using a labile chemical bond that hydrolyzes in vivo, freeing the fully bioactive parent drug, improves solubility of the drug in the bloodstream and permits delivery of the drug to the CNS.
The prodrugs of the present invention exhibit the following useful properties:
The inactive prodrug form helps to mitigate administration-related toxicity;
A therapeutically significant amount of free drug reaches the CNS;
It is expected that the prodrug can be delivered to the CNS via the oral route;
The prodrug is more readily formulated in hydrophilic formulations;
The half-life of elimination of etoposide is extended; and
The conjugate has improved ability to pass from the intestine into the bloodstream.
Etoposide is a member of the epipodophyllotoxin class of compounds and is active against a broad spectrum of tumor types in vitro, including gliomas and astrocytomas (Giaccone, Gazdar et al. 1992; Kasahara, Fujiwara et al. 1992; Brown, McPherson et al. 1995; Chresta, Masters et al. 1996; Beauchesne, Bertrand et al. 1998). Etoposide has a molecular weight of 589 daltons, is lipophilic and is nearly insoluble in water (Hande 1998). As a result, it is typically formulated in a mixture of benzyl alcohol, polysorbate 80/Tween 80, polyethylene glycol 300 and ethanol (VePesid(copyright) Bristol Myers Squibb). For administration VePesid is diluted in physiologically compatible solutions to 0.2 mg/mL to 0.4 mg/mL. Dose levels of 100 mg/m2 to 600 mg/m2 infused intravenously require volumes of 0.4 L to 5.1 L.
Although etoposide is hydrophobic, it does not readily penetrate the blood-brain barrier. As a result, concentrations of etoposide in brain parenchyma remain low after systemic administration (Hande, Wedlund et al. 1984; Donelli, Zucchetti et al. 1992; Kiya, Uozumi et al. 1992). Additionally, while etoposide can reach brain tumors, the concentration of etoposide in the tumor and surrounding tissue remain subtherapeutic (Donelli, Zucchetti et al. 1992). Due to the invasive nature of gliomas and astrocytomas and the difficulty of surgical resection, there is an urgent need for means for delivering drugs, such as etoposide, to areas of the brain that may harbor residual tumor cells.
One way to overcome the hydrophobicity of etoposide is to chemically modify the parent compound to increase its hydrophilicity. However, often such modifications destroy the desired biologic activity. Accordingly, there is a need in the art for amphiphilic etoposide prodrugs which increase penetration of the prodrug across the BBB and to thereby provide active etoposide to the CNS. Additionally, there is a need for a prodrug which facilitates delivery of etoposide into the CNS by other routes of administration, including oral administration. In rodent biodistribution studies, I.V. etoposide is found at significant concentrations in most non-CNS tissues, especially liver and kidney. However, its accumulation in the CNS is very low (Hande, Wedlund et al. 1984; Donelli, Zucchetti et al. 1992). The hydrophobicity of etoposide makes it a good substrate for the P-glycoprotein multidrug pump or related drug transporters found in the endothelium of CNS blood vessels (Schinkel, Smit et al. 1994; Schinkel, Wagenaar et al. 1996). Experimental evidence suggests that these drug transporters act as the element of the blood-brain barrier responsible for actively barring etoposide from the CNS (Abe, Hasegawa et al. 1994). These drug transporters appear also to be partly responsible for clearance of etoposide and other drugs from the blood via direct extrusion through the intestinal wall into the lumen (Leu and Huang 1995; Mayer, Wagenaar et al. 1997; Sparreboom, van Asperen et al. 1997). There is an urgent need in the art for etoposide prodrugs which have been modified to permit the delivery of therapeutic concentrations of etoposide to the CNS by disabling export of the prodrugs by the P-glycoprotein multidrug pump and related drug transporting of the CNS epithelium.
A considerable amount of effort has been devoted to developing extended dosing regimens for etoposide (Greco, Johnson et al. 1991; Hande 1998). These efforts were based on clinical studies demonstrating the superior response rates obtained with 3 to 5 day schedules of administration versus 1 day (Cavalli, Sonntag et al. 1978). Accordingly, there is a need in the art for prodrug versions of the etoposide which extend the metabolic half-life of etoposide.
Etoposide has been shown to be effective in vitro at concentrations as low as 0.1 g/mL (Kasahara, Fujiwara et al. 1992). However, removal of etoposide reverses its inhibition of topoisomerase II, allowing cells to recover (Joel 1996). In addition, etoposide""s effect on topoisomerase II is toxic primarily in the G2 phase of the cell cycle. As a result, a relatively brief treatment with etoposide will kill cells in G2 but allow cells in other phases of the cell cycle to recover. Accordingly, there is a need in the art for etoposide prodrugs which permit chronic dosing, thereby extending treatment of a cycling cell population and leading to cell death of a much greater proportion of the population.
Due to the relatively brief half-life of etoposide, daily (or more often) I.V. infusions are needed to maintain plasma etoposide concentrations in the therapeutic range. Thus, there is a need for etoposide prodrugs which permit an oral dosing regimen with its convenience and lower cost. As noted above, studies with oral etoposide have determined that its bioavailability is erratic, leaving a physician with little assurance that the adequate dose had been delivered. Accordingly, a more consistently bioavailable etoposide prodrug is needed.
In summary, there is a need in the art for a means for permitting producing consistent bioavailability of drugs, such as etoposide, without the necessity of multiple, prolonged I.V. infusions. Furthermore, there is a need for prodrugs which provide anti-cancer agents, such as etoposide, with a sufficiently extended plasma half-life to maintain in vivo concentration in a therapeutic range. Finally, there is a need in the art for prodrugs which enable effective oral delivery of drugs, such as etoposide, and which enables such orally delivered prodrugs to cross the BBB to enter the CNS.
Researchers have modified the physiochemical and pharmacologic properties etoposide by covalently attaching chemical moieties. Etoposide phosphate (Etopophos(copyright), Bristol Myers Squibb) contains a phosphate group at the 4xe2x80x2-position of etoposide, resulting in a prodrug with increased aqueous solubility. The phosphate group hydrolyzes rapidly in vivo, and the compound has the same pharmacologic profile as etoposide. The improved water solubility of this etoposide analog increases the convenience of intravenous infusion; however, the oral bioavailability is only slightly improved, and the variability in bioavailability is still high, presumably because etoposide phosphate is converted into etoposide rapidly in the gut.
Several researchers have pursued formulation of cytotoxic drugs such as paclitaxel, adriamycin and doxorubicin in liposomal or micellar form. One objective of these strategies has been to create xe2x80x9csustained releasexe2x80x9d etoposide, producing extended periods of plasma etoposide concentrations in the therapeutic range. A second objective has been to sequester drugs in these vehicles to restrict access to non-target tissues. However, due to the large size of these particles, it is unlikely that this approach will facilitate improved oral bioavailability or penetration of the BBB. Accordingly, a need remains for etoposide prodrugs which facilitate improved oral bioavailability as well as penetration of the BBB.
A third approach is to conjugate the drug molecule to hydrophilic polymers to improve its solubility. Both polyethylene glycol (PEG) and polyglutamate have been used as hydrophilic polymers in this strategy. It appears that both polymers will increase the solubility of etoposide and therefore improve its handling characteristics. However, the conjugated polymers have been relatively large, resulting in etoposide prodrugs with altered pharmacokinetics and low drug loading. There is therefore a need in the art for etoposide prodrugs with smaller polymers, which prodrugs have improved solubility characteristics as well as the other advantageous attributes described above.
The present invention generally provides amphiphilic prodrugs comprising a drug joined by hydrolyzable bond(s) to one or more straight or branched PEG-oligomer(s) having from 1 to 25 polyethylene glycol units. The PEG-oligomer preferably comprises a salt-forming moiety, which is preferably selected from the group consisting of ammonium and carboxylate. The PEG-oligomers are preferably joined to the therapeutic compound by a bond, such as ester or carbonate, which is hydrolyzable in vivo.
In another aspect of the present invention, the drug portion of the amphiphilic prodrug is etoposide or an etoposide analogue which retains some or all of the therapeutic activity of etoposide.
The amphiphilic prodrug of claim may be derivatized by as many PEG-oligomers as there are sites on the drug for attachment of such PEG-oligomers. Thus, for example, etoposide has 3 sites and can therefore be derivatized by 1, 2 or 3 of the PEG-oligomers.
In another aspect, the amphiphilic prodrugs can be delivered via the oral route of administration to provide a therapeutically effective dose of the drug to the bloodstream. Furthermore, in another aspect, the orally delivered derivatives can provide a therapeutically effective dose of the therapeutic compound to the CNS.
The present invention also provides pharmaceutical compositions comprising the amphiphilic prodrugs of the invention in association with a pharmaceutically acceptable carrier. Such pharmaceutical compositions may be formulated so as to be suitable for oral administration, and may be in any of a wide variety of dosage forms known in the art, such as tablets, capsules, caplets, gelcaps, pills, liquid solutions, suspensions or elixirs, powders, lozenges, micronized particles and osmotic delivery systems.
In another aspect, the present invention provides a amphiphilic prodrug comprising a drug joined by hydrolyzable bond(s) to one or more PEG-oligomer(s) selected from the group consisting of any of the oligomers of Formulae 1-11.
Any of the foregoing oligomers of Formulae 1-11 may also suitably comprise a salt-forming moiety. Preferred salt-forming moieties are ammonium and carboxylate. The preferred drugs are etoposide and etoposide analogues which retain some or all of the therapeutic activity of etoposide, or which exhibit improved activity as compared to etoposide. The drug is derivatized by the number of PEG-oligomers which does not exceed the number of sites of attachment for such PEG-oligomers. Thus where the amphiphilic prodrug is etoposide, it can be derivatized by 1, 2 or 3 of the PEG-oligomers of Formulae 1-11.
The present invention also provides pharmaceutical compositions comprising a amphiphilic prodrug of Formulae 1-11 and a pharmaceutically acceptable carrier. The pharmaceutical composition can be formulated to be suitable for oral administration, and can be in any of a variety of pharmaceutical dosage forms, such as tablets, capsules, caplets, gelcaps, pills, liquid solutions, suspensions or elixirs, powders, lozenges, micronized particles and osmotic delivery systems.
The present invention also provides a method for treating a mammalian subject having a etoposide-responsive disease condition comprising administering to the patient therapeutically effective amount of a amphiphilic prodrug comprising at least one therapeutic compound; and one or more PEG polymers and/or oligomers, each joined to the therapeutic compound by a hydrolyzable bond, said PEG polymers and/or oligomers each comprising one or more straight or branched PEG segments consisting of 2 to 25 polyethylene glycol units. The oligomers optionally comprise a salt-forming moiety.
The present invention also provides a method for treating a mammalian subject having a etoposide-responsive disease condition comprising administering to the patient of therapeutically effective amount of a drug derivativized by any of the Formulae 1-11.
In one aspect of the methods of treatment, the amphiphilic prodrug is delivered via an oral route of administration to provide a therapeutically effective dose of the drug to the bloodstream. In another aspect, the amphiphilic prodrug is delivered via a parenteral route of administration and provides a therapeutically effective dose of the drug to the brain. In yet another aspect, the amphiphilic prodrug is delivered via an oral route of administration and provides a therapeutically effective dose of the drug to the brain. Furthermore, the amphiphilic prodrug may be administered in association with a pharmaceutically acceptable carrier.
In a further aspect, diseases treated according to the therapeutic methods of the invention include cancers, tumors and malignancies, such as acute myelocytic leukemia, bladder carcinoma, breast cancer, cancers of the gastrointestinal tract, Ewing""s sarcoma, Hodgkin""s lymphomas, Kaposi""s sarcoma, leukemias, lung carcinoma, lymphomas, non-Hodgkin""s lymphomas, ovarian cancer, small cell lung cancers and testicular carcinoma.
As used herein the terms xe2x80x9cPEGxe2x80x9d refers to straight or branched polyethylene glycol polymers and monomers. The term xe2x80x9cPEG-oligomerxe2x80x9d refers to oligomers comprising PEG polymers which have been modified to include groups which do not eliminate the amphiphilic properties of such polymers, e.g., without limitation, alkyl, aryl, amino-alkyl and amino-aryl; see also Formulae 2-11 set forth in Section 4, xe2x80x9cDetailed Description of the Invention.xe2x80x9d The term xe2x80x9cPEG subunitxe2x80x9d or xe2x80x9cPEG unitxe2x80x9d refers to a single polyethylene glycol unit, i.e.: xe2x80x94(CH2CH2O)xe2x80x94. The term xe2x80x9cPEG oligomer/polymerxe2x80x9d as used herein refers to PEG oligomers and/or PEG polymers.
As used herein terms such as xe2x80x9cnon-hydrolyzablexe2x80x9d and phrases such as xe2x80x9cnot hydrolyzablexe2x80x9d are used to refer to bonds which cannot be hydrolyzed under normal physiological conditions, as well as bonds which are not quickly hydrolyzed under normal physiological conditions, such as carbamate and amide bonds. The term xe2x80x9chydrolyzablexe2x80x9d refers to bonds which are hydrolyzed under normal physiological conditions, such as ester and carbonate bonds.
A xe2x80x9ctherapeutically effectivexe2x80x9d amount or dose is an amount or dose which prevents, delays or reduces the severity of the onset of disease or other adverse medical condition, or an amount necessary to arrest or reduce the severity of an ongoing disease or other adverse medical condition, and also includes an amount necessary to enhance normal physiological functioning.
As used herein, xe2x80x9cpharmaceutically acceptablexe2x80x9d component (such as a salt, carrier, excipient or diluent) of a formulation according to the present invention is a component which (1) is compatible with the other ingredients of the formulation in that it can be combined with the prodrugs of the present invention without eliminating the biological activity of the prodrugs; and (2) is suitable for use in animals (including humans) without undue adverse side effects (such as toxicity, irritation, and allergic response). Side effects are xe2x80x9cunduexe2x80x9d when their risk outweighs the benefit provided by the pharmaceutical composition. Examples of pharmaceutically acceptable components include, without limitation, any of the standard pharmaceutical carriers such as phosphate buffered saline solutions, water, emulsions such as oil/water emulsion, microemulsions and various types of wetting agents.
FIG. 1. Structure of etoposide showing reactive hydroxyl groups that can be used as sites of attachment for the oligomers of the present invention.
FIG. 2. General scheme of the operation of the present invention. The PEG-oligomer/polymer is attached to the hydroxyl groups on etoposide via a labile ester bond. In the bloodstream, the hydrolyzable bond is cleaved, thereby freeing the active parent compound.
FIG. 3. Cleavage of conjugates in an aqueous environment. Samples of purified etoposide conjugate in phosphate buffers show increasing rate of hydrolysis as the pH increases. At pH 5.9 approximately 10% of the conjugate is hydrolyzed in 28 hours, while at pH 7.4 approximately 40% is hydrolyzed in the same time period. At pH 8.0 hydrolysis is essentially complete after 20 hours. No significant side products were observed during this conversion.
FIG. 4. Graph showing that when the etoposide conjugate was incubated in fresh rat plasma at 37xc2x0 C. hydrolysis of the conjugated etoposide and appearance of free etoposide showed reciprocal behavior, indicating that hydrolysis of the PEG-oligomer/polymer etoposide conjugate is complete and releases free, active etoposide.
FIG. 5. Graph showing in vivo profile of rats given etoposide conjugate versus rats given etoposide. Conjugates of etoposide were administered I.V. to Sprague Dawley rats at 9 xcexcmol/kg. Measurement of free etoposide in plasma at various time points demonstrated that peak plasma etoposide concentration was increased by 60% and the half-life was extended by approximately 50% in rats given the etoposide conjugate (curve I) versus rats given etoposide (curve II).
FIG. 6. When the etoposide concentration in brain tissue was measured in these rats the data revealed that injection with the etoposide conjugate led to a 3-fold increase in accumulation of free etoposide in the brain parenchyma, demonstrating an increased penetration of the blood-brain barrier by conjugated peptide hormones.